The Involvement Of The Mobilization And Asymmetric Division Of Bone Marrow C-kit+Cells In Cardiac Repair After Acute Myocardial Infarciton

Background:Acute myocardial infarction (AMI) is the necrosis of cardio-myocytes causedby the acute and continuous ischemia of coronary artery. It is most common inEurope and America, and in America there are1.5million cases of AMI annually,while, in recent years in China the incidence of AMI has an increase to0.5million ayear, and till now, the total number of the cases has reached to2million, which hasgreatly affected people’s living quality. After acute myocardial infarction (AMI),cardiomyocytes in the AMI-effected region lose their functionality and heartpumping function is severly damaged. If the cardio-myocytes remained had noenough time to reconstitute, heart failur would happen. Recent studies showed that,cardiac stem cells (CSCs c-kit+cells) were discovered to have the function of cardiacrepair. Whereas, for the limitation in number, CSCs are disabled to totally repair theheart after AMI. In cosequence, bone marrow derived c-kit+stem cells became abrand-new chioce. It has been reported that this type of stem cells are able tomobilize to cardiac infarcted regions and perform cardiac repair. Moreover, somecertain progress has been made in some relevant clinically experiments that c-kit+stem cells can repair inrcted cardio-myocytes through the way of cardio-myocytesinjection and inner-vascular injection. However, the motivation of BM c-kit+stemcells after AMI and its mechanism undermine remains unclear. Therefore, we carriedthe present studies to test the motivation and the expression of the c-kit+stem cells inBM-MNCs after AMI, the proliferation capabiliy of the cells and the asymmetric cell divison (ACD) of c-kit+stem cells, aiming to discuss the cardiac repair function ofthem, which provides new idea and support for stem cell therapy.Objectives and Subjects:1.To test the mRNA level of BM c-kit+stem cells after AMI by PCR, and thedynamic rate change of c-kit+stem cells (2、3、7days after operation) using FlowCytometry (FCM) and Magnetic Activated Cell Sorting (MACS), then to directobserve the cells, so as to explore the motivation of BM c-kit+stem cells after AMI.2.Utilizing the Immuno-fluorescent (IF) Staining and the Statistical Analysis todetect the expression level of proliferation related protein ki67and PH3inBM-MNCs and cell subpopulation (c-kit+stem cells and c-kit-cells), and to discussthe active cell proliferation state and its relation with the increase percentage ofc-kit+stem cells in response to AMI.3. Using the similar methods to analyze the asymmetric cell division in the AMImouse BM-MNCs and compare the former with neonatal mouse BM-MNCs, and toinvestigate the prolifration and the cell divisoin pattern of the c-kit+stem cells andc-kit-cells, with the purpose of discussing the possible function of the ACD of c-kit+stem cells in cardiac repair.4.To simply study the ACD phenomenon of BM c-kit+stem cells and c-kit-cellsin patients with Coronary Heart Disease, and its relative percentage, and to discoverthe possible increase of ACD proportion in human BM c-kit+dividing cells inresponse to heart ischemic injury.Methods：Male C57BL/6wild-type mice at8-10weeks of age (22-25g) were used andgrouped randomly. The mice in AMI group were anesthetized with gaseousisoflurane and then the left anterior descending coronary artery was tightened with asuture after thoracotomy. The control group underwent the same surgical procedurewithout coronary ligation.2、3and7days after AMI, mice were sacrificed and BM-MNCs were collected. First of all, PCR、FCM and MACS were utilized todetect and statistically analyze the dynamic change of c-kit+stem cells percentage inpost-infarct BM, and to sort out the c-kit+stem cells and c-kit-cells. Here,Immuno-fluorescent (IF) Staining was used to visually confirm the positive cells.Next, the proliferation and division state of BM-MNCs were investigated using theki67and PH3and analyzed statistically, and the proliferation standard of BM cellsubpopulation (c-kit+stem cells and c-kit-cells) were compared, aiming to clarify therelation between the increase and the proliferation level of c-kit+stem cells. ThenNumb and α-adaptin was IF stained respectively and the ACD phenomena weretested and compared between the adult mouse BM-MNCs and neonatal mouseBM-MNCs. Finally, Numb was chosen to compare the ACD in the c-kit+stem cellsand c-kit-cells and to statistically analyze the differences.Results:1. The identification and dynamic change of c-kit+cells in the mouseBM-MNCs in response to AMI:(1) The PCR results showed that the c-kit RNA level in the AMI BM-MNCs ismuch higher than the control group, and day3is higher than that of day7.(2) FCM results demonstrated that the percentage of c-kit+stem cells after AMIincreased, and AMI group as follows:6.98±1.18%、17.13±1.5%、5.95±0.68%, andhas a significant difference compared with the control group (4.47±0.88%);3daysafter AMI the percentage reached the maximum and had a marked differencecompared with that of day2and7(p <0.05).(3) The MACS results showed a similar dynamic trend of c-kit+stem cellspercentage with FCM.(4) IF staining confirmed the direct existence of the c-kit+stem cells inBM-MNCs2. The comparison of proliferation rate among the BM-MNCs, c-kit+stem cells and c-kit-cells after AMI:(1) IF results confirmed the proliferation ability of BM-MNCs on AMI day3and7, and the statistical methods showed that the ki67and PH3level were bothhigher in the AMI group than the control group (p <0.05).(2) The ki67standard of BM c-kit+stem cells was higher than the negative cellsfor AMI day3(8.34±0.25%vs1.90±0.34%，p <0.05).(3) The PH3level of the cell subpopulation was similar (5.98±0.34%vs2.26±0.40%，p <0.05).3. The occurrence and the comparison of ACD in the adult and neonatal mouseBM-MNCs3days after AMI:(1) In adult and neonatal mouse BM-MNCs, the IF stained Numb wasdiscovered to be distributed to one pole of the dividing cells; and statistical datashowed that ACD proportion of the former two cell group had no significantdifferences (11.7±1.25%and9.33±0.94%), however both higher than the controlgroup (p <0.05).(2) The IF staining results of alpha-adaptin illustrated that the asymmetricdistribution of cell fate determinate, the percentage of which is9.41±1.26%and8.33±0.76%respectively. Similarly, the percentage of two groups had no significantdifferences, and both higher than the control group (p <0.05).4. ACD preferentially occurs in the BM c-kit+stem cells after acute myocardialinfarction, but not in c-kit-cellsIt was discovered that cell fate determinate Numb was shown to be distributedto one polar of the cell, and in c-kit+stem cells and c-kit-cells the percentage ofACD go to12.57±1.39%and4.17±1.65%, and they had distinct difference (p<0.05).5. The asymmetric cell division in BM c-kit+and c-kit-cells derived from humanwith Coronary Heart Disease (CHD) BM c-kit+stem cells were sorted by MACS and10%of BM-MNCs werediscovered c-kit postive; and the asymmetric Numb distribution was discovered tobe14.29%and6.06%for c-kit+and c-kit-cells.Conclusions:1. BM was stimulated by ischemic injury and c-kit+stem cells percentageincreased in response to AMI and the cells were motivated to the heart to performcardiac repair; day3is most active time point in response to AMI.2. It is mainly the c-kit+stem cells in the BM-MNCs that exert activeproliferation and division, then motivated the BM cells to perform cardiac repair inresponse to AMI, not the c-kit-cells.3. Duing to the stimuli of the cardiac ischemic injury after AMI, mouseBM-MNCs performed ACD and possibly went back to the vigorous state of activeproliferation and division.4. In response to the cardiac injury, it is the c-kit+stem cells that were stimulatedto utilize ACD to exert self-renewal and differentiation, which maintained the stemcell number and produced differentiated cells to perform cardiac repair.5. In the BM of Coronary Heart Disease patients, the c-kit+stem cells weremotivated after ischemic injury and performed ACD phenomenon, which may berelevant to the cardiac repair.